The amplification of nucleic acids is useful in a variety of applications. For example, nucleic acid amplification methods have been used in the identification of genetic disorders such as sickle-cell anemia and cystic fibrosis, in detecting the presence of infectious organisms, and in typing and quantification of DNA and RNA for cloning and sequencing.
Methods of amplifying nucleic acid sequences are known in the art. One method, known as the polymerase chain reaction ("PCR"), utilizes a pair of oligonucleotide sequences called "primers" and thermal cycling techniques wherein one cycle of denaturation, annealing, and primer extension results in a doubling of the target nucleic acid of interest. PCR amplification is described further in U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference.
Another method of amplifying nucleic acid sequences known in the art is the ligase chain reaction ("LCR"). Like PCR, LCR utilizes thermal cycling techniques. In LCR, however, two primary probes and two secondary probes are employed instead of the primer pairs used in PCR. By repeated cycles of hybridization and ligation, amplification of the target is achieved. The ligated amplification products are functionally equivalent to either the target nucleic acid or its complement. This technique is described more completely in EP-A-320 308 and EP-A-439 182.
Other methods of amplifying nucleic acids known in the art involve isothermal reactions, including the reaction referred to as Q-beta ("Q.beta.") amplification [See, for example, Kramer et al., U.S. Pat. No. 4,786,600, WO 91/04340, Cahill et al., Clin. Chem., 37:1482-1485 (1991); Pritchard et al., Ann. Biol. Clin., 48:492-497 (1990)]. Another isothermal reaction is described in Walker et al., "Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system", Proc. Natl. Acad. Sci., 89:392-396 (1992). These amplification reactions do not require thermal cycling.
Amplification of nucleic acids using such methods is usually performed in a closed reaction vessel such as a snap-top vial. After the amplification, the reaction vessel is then opened and the amplified product is transferred to a detection apparatus where standard detection methodologies are used.
In some cases, the amplified product is detected by denaturing the double-stranded amplification products, and treating those products with one or more hybridizing probes having a detectable label. The unhybridized probes are typically separated from the hybridized probe, requiring an extra separation step. Alternatively, the primer or probes may be labeled with a hapten as a reporter group. Following amplification, the hapten, which has been incorporated into the amplification product, can be used for separation and/or detection.
In yet another detection method, the amplification products may be detected by gels stained with ethidium bromide. In sum, .sup.32 P tracings, enzyme immunoassay [Keller et al., J. Clin. Microbiology, 28:1411-6 (1990)], fluorescence [Urdea et al., Nucleic Acids Research, 16:4937-56 (1988); Smith et at., Nucleic Acids Research, 13:2399-412 (1985)], and chemiluminescence assays and the like can be performed to detect nucleic acids in a heterogeneous manner [Bornstein and Voyta, Clin. Chem., 35:1856-57 (1989); Bornstein et al., Anal. Biochem., 180:95-98 (1989); Tizard et al., Proc. Natl. Acad. Sci., 78:4515-18 (1990)] or homogeneous manner [Arnold et al., U.S. Pat. No. 4,950,613; Arnold et al., Clin. Chem., 35:1588-1589 (1989); Nelson and Kacian, Clinica Chimica Acta, 194:73-90 (1990)].
In each case, however, these detection procedures have serious disadvantages. First, when the reaction vessel containing a relatively high concentration of the amplified product is opened, a splash or aerosol is usually formed. Such a splash or aerosol can be sources of potential contamination, and contamination of negative, or not-yet amplified, nucleic acids is a serious problem and may lead to erroneous results.
Similar problems concerning contamination may involve the work areas and equipment used for sample preparation, preparation of the reaction reagents, amplification, and analysis of the reaction products. Such contamination may also occur through contact transfer (carryover), or by aerosol generation.
Furthermore, these previously described detection procedures are time-consuming and labor intensive. In the case of both hybridization probes and hapten detection, the amplification reaction vessel must be opened and the contents transferred to another vessel, medium or instrument. Such an "open" detection system is disadvantageous as it leads to further contamination problems, both airborne and carryover.
Thus, a need emerges for detecting amplified nucleic acids in a closed system in order to eliminate the potential for contamination. Also, a need emerges for a method of amplifying and detecting the target nucleic acid in an operationally simple, yet highly sensitive manner. The ability to detect the amplification product in a sealed vessel, or in a closed system, offers useful advantages over existing prior art methods, including the ability to monitor the amplification of target nucleic acid throughout the course of the reaction.
The use of total internal reflection fluorescence techniques is known in the art with respect to immunoassays [Harrick, et al., Anal. Chem., 45:687 (1973)]. Devices and methods that use total internal reflection fluorescence for immunoassays have been described in the art by Hirschfield, U.S. Pat. Nos. 4,447,564, 4,577,109, and 4,654,532; Hirschfield and Block, U.S. Pat. Nos. 4,716,121 and 4,582,809, which are all incorporated herein by reference. Other descriptions and uses are given by Glass, U.S. Pat. No. 4,844,869; Andarde, U.S. Pat. No. 4,368,047; Hirschfield, GB 2,190,189A; Lackie, WO 90/067,229; Block, GB 2,235,292A, and Carter et al., U.S. Pat. No. 4,608,344.
Use of total internal reflection elements allows performing a homogeneous assay (i.e. free of separation and wash steps) for members of specific binding pairs. Several applications of this principle are known in the art [such as Kronick, et al. J. Immunol. Methods, 8:235 (1975) and U.S. Pat. No. 3,604,927] for hapten assays and for immunoassay of macromolecules [Sutherland et al., J. Immunol. Methods, 74:253 (1984)].
In known total internal reflectance methods, however, the slow diffusion of members of specific binding pairs from the bulk of the solution to the surface of the TIR element creates a limitation in using TIR fluorescence techniques. Thus, prior art devices have used capillary tubes or flow cells to enhance diffusion either by limiting the diffusion distances or by continuous exposure to fresh reactant stream, or both. But these systems, too, have drawbacks that make them less than optimal for clinical biological applications. Capillary tubes are difficult to manipulate and are not easily automated. Flow cells require extensive washing in an effort to reduce carryover contamination before they can be reused.
Thus, in addition to a need for contamination-free, closed amplification systems, there is also a need in the art for better TIR assay systems that are more easily automated and even disposable if desired.